Japanese Encephalitis Virus prM Research Articles

The GTPase activity and isoprenylation of Swine GBP1 are critical for inhibiting the production of Japanese Encephalitis Virus

Japanese encephalitis virus (JEV) is a flavivirus that cause severe neurological deficits. The guanylate-binding protein 1 (GBP1) gene is an interferon-stimulated gene and exerts antiviral functions on many RNA and DNA viruses via diverse mechanisms, however, the roles and the action modes of GBP1 in the antiviral effect on the production of JEV RNA and infectious virions remain to be clarified. In this study, we found that the RNA levels of swine GBP1 (sGBP1) in PK15 cells were up-regulated at the late stage of JEV infection. The overexpression of sGBP1 significantly inhibited the production of JEV while the knockdown of sGBP1 promoted the production of JEV. The GTPase activity and isoprenylation of sGBP1 both are critical for anti-JEV activity. The GTPase activity of sGBP1 is responsible for inhibiting the production of JEV genomic RNA. The isoprenylation of sGBP1 inhibited the expression and cleavage of JEV prM to decrease the yields of infectious virions, which may be associated with the interaction between sGBP1 and cellular proprotein convertase furin. Taken together, the study dissected the action modes of sGBP1with potent anti-JEV activity in more details.

Flavivirus prM interacts with MDA5 and MAVS to inhibit RLR antiviral signaling

BackgroundVector-borne flaviviruses, including tick-borne encephalitis virus (TBEV), Zika virus (ZIKV), West Nile virus (WNV), yellow fever virus (YFV), dengue virus (DENV), and Japanese encephalitis virus (JEV), pose a growing threat to public health worldwide, and have evolved complex mechanisms to overcome host antiviral innate immunity. However, the underlying mechanisms of flavivirus structural proteins to evade host immune response remain elusive.ResultsWe showed that TBEV structural protein, pre-membrane (prM) protein, could inhibit type I interferon (IFN-I) production. Mechanically, TBEV prM interacted with both MDA5 and MAVS and interfered with the formation of MDA5-MAVS complex, thereby impeding the nuclear translocation and dimerization of IRF3 to inhibit RLR antiviral signaling. ZIKV and WNV prM was also demonstrated to interact with both MDA5 and MAVS, while dengue virus serotype 2 (DENV2) and YFV prM associated only with MDA5 or MAVS to suppress IFN-I production. In contrast, JEV prM could not suppress IFN-I production. Overexpression of TBEV and ZIKV prM significantly promoted the replication of TBEV and Sendai virus.ConclusionOur findings reveal the immune evasion mechanisms of flavivirus prM, which may contribute to understanding flavivirus pathogenicity, therapeutic intervention and vaccine development.

Enhancement of autophagy as a strategy for development of new DNA vaccine candidates against Japanese encephalitis

For decades, an on-going concerted effort has been made to develop a universal DNA vaccine to combat the looming threat of a potential outbreak of the emerging Japanese encephalitis virus (JEV) infection. However, effective strategies are urgently required to counter poor immunogenicity and insufficient long-term protection. Recent reports have confirmed the critical role of autophagy in antigen presentation, long-term immune memory and immune responses against JEV. In this study, JEV prM and E protein with strong immunogenicity were fused with microtubule-associated protein 1 light chain 3 (LC3) encoding gene to construct an autophagy-mediated pJME-LC3 DNA vaccine. Researches indicated significant increase of autophagosomes or LC3 Ⅱ expression in pJME-LC3 transfected cells. Furthermore, prME-LC3 fused protein was observed co-localized with GFP-LC3 to autophagosomes, which means it was successfully targeted to autophagosomes. After immunizing with pJME-LC3, mice were detected highest proportion of CD3+CD8+ T lymphocytes, CD8+ effector memory T cells (TEMs) and JEV specific cytotoxic T lymphocyte (CTL) activity to eliminate JEV. pJME-LC3 also enhanced IgG2a antibody in serum and cytokines IFN-γ, IL-12 produced by splenocytes, thus skew toward Th1 type immune response by activating the JAK2/STAT1 signaling pathway and upregulating expression of transcription factor T-bet. Notably, mice immunized with pJME-LC3 showed highest survival rate and long-lasting neutralizing antibody when challenged with virulent JEV, which were consistent with augment in percentage of CD4+ central memory T cells (TCMs). In brief, our studies suggested that autophagy can be used as a optimization strategy to enhance JEV specific immune response and long-term immune memory. Our attempt will contribute towards future efforts to develop an efficacious JEV vaccine.

A Japanese Encephalitis Virus Vaccine Inducing Antibodies Strongly Enhancing In Vitro Infection Is Protective in Pigs.

The Japanese encephalitis virus (JEV) is responsible for zoonotic severe viral encephalitis transmitted by Culex mosquitoes. Although birds are reservoirs, pigs play a role as amplifying hosts, and are affected in particular through reproductive failure. Here, we show that a lentiviral JEV vector, expressing JEV prM and E proteins (TRIP/JEV.prME), but not JEV infection induces strong antibody-dependent enhancement (ADE) activities for infection of macrophages. Such antibodies strongly promoted infection via Fc receptors. ADE was found at both neutralizing and non-neutralizing serum dilutions. Nevertheless, in vivo JEV challenge of pigs demonstrated comparable protection induced by the TRIP/JEV.prME vaccine or heterologous JEV infection. Thus, either ADE antibodies cause no harm in the presence of neutralizing antibodies or may even have protective effects in vivo in pigs. Additionally, we found that both pre-infected and vaccinated pigs were not fully protected as low levels of viral RNA were found in lymphoid and nervous system tissue in some animals. Strikingly, the virus from the pre-infection persisted in the tonsils throughout the experiment. Finally, despite the vaccination challenge, viral RNA was detected in the oronasal swabs in all vaccinated pigs. These latter data are relevant when JEV vaccination is employed in pigs.

Construction and immune efficacy of recombinant pseudorabies virus expressing PrM-E proteins of Japanese encephalitis virus genotype І.

BackgroundJapanese encephalitis (JE) is an arboviral disease with high case fatality rates and neurologic or psychiatric sequelae among survivors in Asia, western Pacific countries and northern Australia. Japanese encephalitis virus (JEV) is the cause of JE and the emergence of genotype І (GI) JEV has displaced genotype III (GIII) as the dominant strains circulating in some Asian regions. The currently available JE vaccines are safe and effective in preventing this disease, but they are developed based on the GIII JEV strains.MethodsThe recombinant virus PRV TK−/gE−/PrM-E+ which expressed the premembrane (prM) and envelope (E) proteins of JEV SX09S-01 strain (genotype I, GI) was constructed by homologous recombination between the genome of PRV TK−/gE−/LacZ+ digested with EcoRI and plasmid pIE-CAG-PrM-E-BGH. Expression of JEV PrM and E proteins was analyzed by Western blot analysis. Immune efficacy of PRV TK−/gE−/PrM-E+ was further evaluated in mouse model.ResultsA recombinant pseudorabies virus (PRV TK−/gE−/PrM-E+) was successfully constructed. Mice experiments showed that PRV TK−/gE−/PrM-E+ could induce a high level of ELISA antibodies against PRV and JEV, as well as high titer of PRV neutralizing antibodies. After challenge with 1 × 107 PFU virulent JEV SX09S-01 strain, the time of death was delayed and the survival rate was improved in PRV TK−/gE−/PrM-E+ vaccinated mice.ConclusionsPRV TK−/gE−/PrM-E+ is a potential vaccine candidate against PRV and JEV GI infection in the future.

Generation and characterization of a new mammalian cell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate.

BackgroundJapanese encephalitis virus (JEV) is the most important cause of epidemic encephalitis in most Asian regions. There is no specific treatment available for Japanese encephalitis, and vaccination is the only effective way to prevent JEV infection in humans and domestic animals. The purpose of this study is to establish a new mammalian cell line stably and efficiently expressing virus-like particle of JEV for potential use of JEV subunit vaccine.ResultsWe generated a new cell clone (BJ-ME cells) that stably produces a secreted form of Japanese encephalitis virus (JEV) virus-like particle (VLP). The BJ-ME cells were engineered by transfecting BHK-21 cells with a code-optimized cDNA encoding JEV prM and E protein expression plasmid. Cell line BJ-ME can stably produces a secreted form of Japanese encephalitis virus virus-like particle (JEV-VLP) which contains the JEV envelope glycoprotein (E) and membrane protein (M). The amount of JEV-VLP antigen released into the culture fluid of BJ-ME cells was as high as 15–20 μg/ml. JEV-VLP production was stable after multiple cell passages and 100% cell expression was maintained without detectable cell fusion or apoptosis. Cell culture fluid containing the JEV-VLP antigen could be harvested five to seven times continuously at intervals of 4–6 days while maintaining the culture. Mice immunized with the JEV-VLP antigen with or without adjuvant developed high titers of neutralizing antibodies and 100% protection against lethal JEV challenge.ConclusionThese results suggest that the recombinant JEV-VLP antigen produced by the BJ-ME cell line is an effective, safe and affordable subunit Japanese encephalitis vaccine candidate, especially for domestic animals such as pig and horse.

Characterization of the GXXXG motif in the first transmembrane segment of Japanese encephalitis virus precursor membrane (prM) protein

The interaction between prM and E proteins in flavivirus-infected cells is a major driving force for the assembly of flavivirus particles. We used site-directed mutagenesis to study the potential role of the transmembrane domains of the prM proteins of Japanese encephalitis virus (JEV) in prM-E heterodimerization as well as subviral particle formation. Alanine insertion scanning mutagenesis within the GXXXG motif in the first transmembrane segment of JEV prM protein affected the prM-E heterodimerization; its specificity was confirmed by replacing the two glycines of the GXXXG motif with alanine, leucine and valine. The GXXXG motif was found to be conserved in the JEV serocomplex viruses but not other flavivirus groups. These mutants with alanine inserted in the two prM transmembrane segments all impaired subviral particle formation in cell cultures. The prM transmembrane domains of JEV may play importation roles in prM-E heterodimerization and viral particle assembly.

Co-expression of Japanese encephalitis virus prM–E–NS1 antigen with granulocyte-macrophage colony-stimulating factor enhances humoral and anti-virus immunity after DNA vaccination

Japanese encephalitis virus (JEV) is an agent of Japanese encephalitis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) is an attractive DNA vaccine adjuvant for its antigen presentation. In the present study, we have constructed DNA vaccines that carried JEV prM–E–NS1 genes with or without the GM-CSF gene. Immunization with the bicistronic plasmid pCAG-JEGM that co-expresses GM-CSF and viral prM–E–NS1, resulted in the highest IgG response and sufficient protection against virus-challenged BALB/c mice. However, much to our surprise, co-inoculation of the GM-CSF plasmid with the pCAG-JE plasmid expressing viral prM–E–NS1 lead to a low antibody titer and a relatively low survival rate. Moreover, anamnestic antibody-mediated protection played a dominant role in the mice JEV challenge model, according to the enhancement of post-challenge neutralizing antibody titers and further adoptive transfer experiments. Taken together, this study should encourage further development of JEV DNA vaccine strategies and caution against the use of cytokines as an adjuvant.

A Plasmid Encoding Japanese Encephalitis Virus PrM and E Proteins Elicits Protective Immunity in Suckling Mice

A plasmid encoding Japanese encephalitis virus (JEV) prM and E proteins was constructed, and its efficacy as a candidate vaccine against JEV was evaluated in suckling mice. Groups of 10 BALB/c mice (5-7 days old) were immunized twice via muscular injection with this DNA vaccine, an empty vector or PBS at an interval of 3 weeks, and were challenged with a lethal dose of JEV 3 weeks after the second inoculation. Both cellular and humoral immune responses were examined before the challenge. Two animals from each group were sacrificed to detect the JEV-specific cytotoxic T lymphocyte activity. JEV-specific lactate dehydrogenase release in the DNA vaccine, empty vector and PBS groups was 37.5%, 18% and 8.5% respectively. JEV-specific antibody was detected in 8 of 10 animals in DNA vaccine group with a geometrical mean titer of 1: 28.3. The pooled serum from the same group also showed a neutralizing activity. Six of 8 mice in the DNA vaccine group survived the challenge, with a protection rate of 75%, but all the mice died in the two control groups. These results show that this JEV prM and E DNA vaccine is immunogenic and protective against JEV infection in the mouse model.

Protection against lethal Japanese encephalitis virus infection of mice by immunization with the highly attenuated MVA strain of vaccinia virus expressing JEV prM and E genes

Genes encoding the glycosylated precursor of the membrane (prM) and envelope (E) proteins of a Korean strain of Japanese encephalitis virus (JEV) were inserted into the genome of the host-range restricted, highly attenuated, and safety-tested MVA strain of vaccinia virus. MVA recombinants containing the JEV genes, under strong synthetic or modified H5 vaccinia virus promoters, were isolated. Synthesis of JEV prM and E proteins was detected by immunofluorescence microscopy, flow cytometry, and polyacrylamide gel electrophoresis. Mice inoculated and boosted by various routes with either of the MVA recombinants produced JEV neutralizing antibodies, that had titres comparable with those induced by an inactivated JEV vaccine, as well as haemagglutination-inhibiting antibodies. Mice immunized with 2×10 6 infectious units of MVA/JEV recombinants by intramuscular or intraperitoneal routes were completely protected against a 10 5 LD 50 JEV challenge at 9 weeks of age.